Candida albicans is an important opportunistic fungal pathogen that causes both mucosal and invasive disease in immunocompromised patients. Nearly all HIV/AIDS patients will develop oropharyngeal (OPC) or esophageal candidiasis during their lifetime. Because of its recurrent nature, the development of drug-resistant OPC/EC is an important problem, leading to current efforts toward developing novel approaches to therapy based on modulation of C. albicans virulence. For such strategies to be successful, an improved understanding of C. albicans pathogenesis is needed. The ability of C. albicans to undergo morphogenesis from yeast to hyphal form is clearly required for the organism to cause disease. However, the regulatory mechanisms that control hypha formation are incompletely defined. In this project, we have developed a novel large scale genetic screening strategy to identify interactions between pairs of genes that affect hyphal development. The strategy is based on a genetic phenomenon called complex haploinsufficiency (CHI) which occurs when heterozygous mutations in two separate genes cause a more severe phenotype than either single mutation by itself. We have used transposon mutagenesis to generate a large set of double mutants starting with a strain lacking one allele of CBK1, a protein kinase involved in hyphal development. Preliminary data indicate that this screen has identified a number of novel genes involved in hyphal development and has provided evidence supporting the hypothesis that CBK1 acts in parallel with the protein kinase A pathway to regulated hyphal development. In this application, we propose to further develop this screen by pursuing the following specific aims: 1) Identify genes that display complex haploinsufficient interactions with CBK1 in C. albicans;2) Test the hypothesis that a CHI screen of CBK1 will identify targets of the Cbk1-dependant transcription factor Ace2;and 3) Test the hypothesis that the RAM (CBK1) and cAMP-PKA signaling pathways function in parallel during hypha development. PUBLIC HEALTH RELEVANCE: Candida albicans is an important opportunistic fungal pathogen that causes disease in immunocompromised patients. Nearly all HIV/AIDS patients will develop oropharyngeal or esophageal candidal infection during their lifetime. The ability of C. albicans to cause disease is directly related to its ability to change its shape from a round yeast form to an elongated filamentous form. The proposed research will probe the genetic mechanisms by which C. albicans undergoes this change. We have designed a novel genetic screening strategy to identify genes involved in filament formation that would not otherwise be apparent from standard screening approaches. The mechanistic information generated by this study should help design new antifungal therapies targeted at processes involved in pathogenesis.